Multinational evaluation of genetic diversity indicators for the Kunming-Montreal Global Biodiversity Framework.

Under the recently adopted Kunming-Montreal Global Biodiversity Framework, 196 Parties committed to reporting the status of genetic diversity for all species. To facilitate reporting, three genetic diversity indicators were developed, two of which focus on processes contributing to genetic diversity conservation: maintaining genetically distinct populations and ensuring populations are large enough to maintain genetic diversity. The major advantage of these indicators is that they can be estimated with or without DNA-based data. However, demonstrating their feasibility requires addressing the methodological challenges of using data gathered from diverse sources, across diverse taxonomic groups, and for countries of varying socio-economic status and biodiversity levels. Here, we assess the genetic indicators for 919 taxa, representing 5271 populations across nine countries, including megadiverse countries and developing economies. Eighty-three percent of the taxa assessed had data available to calculate at least one indicator. Our results show that although the majority of species maintain most populations, 58% of species have populations too small to maintain genetic diversity. Moreover, genetic indicator values suggest that IUCN Red List status and other initiatives fail to assess genetic status, highlighting the critical importance of genetic indicators.

An insight into vitamin E and lipid nutrition of the plains-wanderer Pedionomus torquatus.

Vitamin E, as α-tocopherol, is an essential antioxidant protecting the body from free radicals. The vitamin E requirement of managed wildlife species is known to be greater than their wild counterparts, predominantly due to higher dietary lipid content and potentially stressful environments. The plains-wanderer (Pedionomus torquatus, Family Pedionomidae [monotypical]) is a critically endangered, superficially quail-like bird that is the focus of an ongoing captive breeding programme in Australia. It is estimated that plains-wanderers have a high vitamin E requirement (compared with domestic poultry species) to offset a high lipid diet and their naturally flighty temperament. This study therefore aims to gain a greater understanding of the nutritional status and vitamin E requirements of plains-wanderers in managed environments. Total lipid and α-tocopherol intake were quantified for 26 zoo-managed plains-wanderers over a series of diet intake trials in addition to measurement of plasma α-tocopherol and cholesterol concentrations. Plains-wanderers that consumed higher portions of dietary fat had significantly lower circulating α-tocopherol concentrations than birds that consumed lower total dietary fat (p < .001). Additionally, plasma cholesterol concentrations of managed plains-wanderers were found to be significantly greater than all other bird species reviewed, irrespective of Family or feeding type. We also present the first published data quantifying the nutritional makeup of stomach contents of a wild plains-wanderer for use as a potential guide for diet formulation. This study forms a vital foundational insight into the nutritional management of plains-wanderers, but further research is required to understand their dietary habits and cholesterol metabolism.

Genetic diversity goals and targets have improved, but remain insufficient for clear implementation of the post-2020 global biodiversity framework.

Genetic diversity among and within populations of all species is necessary for people and nature to survive and thrive in a changing world. Over the past three years, commitments for conserving genetic diversity have become more ambitious and specific under the Convention on Biological Diversity's (CBD) draft post-2020 global biodiversity framework (GBF). This Perspective article comments on how goals and targets of the GBF have evolved, the improvements that are still needed, lessons learned from this process, and connections between goals and targets and the actions and reporting that will be needed to maintain, protect, manage and monitor genetic diversity. It is possible and necessary that the GBF strives to maintain genetic diversity within and among populations of all species, to restore genetic connectivity, and to develop national genetic conservation strategies, and to report on these using proposed, feasible indicators.

How much is enough? Sampling intensity influences estimates of reproductive variance in an introduced population.

Conservation introductions to islands and fenced enclosures are increasing as in situ mitigations fail to keep pace with population declines. Few studies consider the potential loss of genetic diversity and increased inbreeding if released individuals breed disproportionately. As funding is limited and post-release monitoring expensive for conservation programs, understanding how sampling effort influences estimates of reproductive variance is useful. To investigate this relationship, we used a well-studied population of Tasmanian devils (Sarcophilus harrisii) introduced to Maria Island, Tasmania, Australia. Pedigree reconstruction based on molecular data revealed high variance in number of offspring per breeder and high proportions of unsuccessful individuals. Computational subsampling of 20%, 40%, 60%, and 80% of observed offspring resulted in inaccurate estimates of reproductive variance compared to the pedigree reconstructed with all sampled individuals. With decreased sampling effort, the proportion of inferred unsuccessful individuals was overestimated and the variance in number of offspring per breeder was underestimated. To accurately estimate reproductive variance, we recommend sampling as many individuals as logistically possible during the early stages of population establishment. Further, we recommend careful selection of colonizing individuals as they may be disproportionately represented in subsequent generations. Within the conservation management context, our results highlight important considerations for sample collection and post-release monitoring during population establishment.

Exploiting genomic synteny in Felidae: cross-species genome alignments and SNV discovery can aid conservation management.

BACKGROUND: While recent advances in genomics has enabled vast improvements in the quantification of genome-wide diversity and the identification of adaptive and deleterious alleles in model species, wildlife and non-model species have largely not reaped the same benefits. This has been attributed to the resources and infrastructure required to develop essential genomic datasets such as reference genomes. In the absence of a high-quality reference genome, cross-species alignments can provide reliable, cost-effective methods for single nucleotide variant (SNV) discovery. Here, we demonstrated the utility of cross-species genome alignment methods in gaining insights into population structure and functional genomic features in cheetah (Acinonyx jubatas), snow leopard (Panthera uncia) and Sumatran tiger (Panthera tigris sumatrae), relative to the domestic cat (Felis catus). RESULTS: Alignment of big cats to the domestic cat reference assembly yielded nearly complete sequence coverage of the reference genome. From this, 38,839,061 variants in cheetah, 15,504,143 in snow leopard and 13,414,953 in Sumatran tiger were discovered and annotated. This method was able to delineate population structure but limited in its ability to adequately detect rare variants. Enrichment analysis of fixed and species-specific SNVs revealed insights into adaptive traits, evolutionary history and the pathogenesis of heritable diseases. CONCLUSIONS: The high degree of synteny among felid genomes enabled the successful application of the domestic cat reference in high-quality SNV detection. The datasets presented here provide a useful resource for future studies into population dynamics, evolutionary history and genetic and disease management of big cats. This cross-species method of variant discovery provides genomic context for identifying annotated gene regions essential to understanding adaptive and deleterious variants that can improve conservation outcomes.

First evidence of deviation from Mendelian proportions in a conservation programme.

Classic Mendelian inheritance is the bedrock of population genetics and underpins pedigree-based management of animal populations. However, assumptions of Mendelian inheritance might not be upheld in conservation breeding programmes if early viability selection occurs, even when efforts are made to equalise genetic contributions of breeders. To test this possibility, we investigated deviations from Mendelian proportions in a captive metapopulation of the endangered Tasmanian devil. This marsupial population is ideal for addressing evolutionary questions in conservation due to its large size, range of enclosure types (varying in environmental conditions), good genomic resources (which aid interpretation), and the species' biology. Devil mothers give birth to more offspring than they can nurse in the pouch, providing the potential for intense viability selection amongst embryos. We used data from 140 known sire-dam-offspring triads to isolate within-family selection from population-level mechanisms (such as mate choice or inbreeding), and compared observed offspring genotypes at 123 targeted SNPs to neutral (i.e., Mendelian) expectations. We found lower offspring heterozygosity than expected, and subtle patterns that varied across a gradient of management intensity from zoo-like enclosures to semi-wild environments for some loci. Meiotic drive or maternal-foetal incompatibilities are consistent with our results, although we cannot statistically confirm these mechanisms. We found some evidence that maternal genotype affects annual litter size, suggesting that family-level patterns are driven by differential offspring mortality before birth or during early development. Our results show that deviations from Mendelian inheritance can occur in conservation programmes, despite best-practice management to prevent selection.

Global Commitments to Conserving and Monitoring Genetic Diversity Are Now Necessary and Feasible.

Global conservation policy and action have largely neglected protecting and monitoring genetic diversity-one of the three main pillars of biodiversity. Genetic diversity (diversity within species) underlies species' adaptation and survival, ecosystem resilience, and societal innovation. The low priority given to genetic diversity has largely been due to knowledge gaps in key areas, including the importance of genetic diversity and the trends in genetic diversity change; the perceived high expense and low availability and the scattered nature of genetic data; and complicated concepts and information that are inaccessible to policymakers. However, numerous recent advances in knowledge, technology, databases, practice, and capacity have now set the stage for better integration of genetic diversity in policy instruments and conservation efforts. We review these developments and explore how they can support improved consideration of genetic diversity in global conservation policy commitments and enable countries to monitor, report on, and take action to maintain or restore genetic diversity.

Moving from trends to benchmarks by using regression tree analysis to find inbreeding thresholds in a critically endangered bird.

Understanding how inbreeding affects endangered species in conservation breeding programs is essential for their recovery. The Hawaiian Crow ('Alala) (Corvus hawaiiensis) is one of the world's most endangered birds. It went extinct in the wild in 2002, and, until recent release efforts starting in 2016, nearly all of the population remained under human care for conservation breeding. Using pedigree inbreeding coefficients (F), we evaluated the effects of inbreeding on Hawaiian Crow offspring survival and reproductive success. We used regression tree analysis to identify the level of inbreeding (i.e., inbreeding threshold) that explains a substantial decrease in 'Alala offspring survival to recruitment. Similar to a previous study of inbreeding in 'Alala, we found that inbreeding had a negative impact on offspring survival but that parental (vs. artificial) egg incubation improved offspring survival to recruitment. Furthermore, we found that inbreeding did not substantially affect offspring reproductive success, based on the assumption that offspring that survive to adulthood breed with distantly related mates. Our novel application of regression tree analysis showed that offspring with inbreeding levels exceeding F = 0.098 were 69% less likely to survive to recruitment than more outbred offspring, providing a specific threshold value for ongoing population management. Our results emphasize the importance of assessing inbreeding depression across all life history stages, confirm the importance of prioritizing parental over artificial egg incubation in avian conservation breeding programs, and demonstrate the utility of regression tree analysis as a tool for identifying inbreeding thresholds, if present, in any pedigree-managed population.

Moverse de las Tendencias a los Referentes Mediante el Análisis de Árbol de Regresión para Encontrar los Umbrales de Endogamia de una Ave en Peligro Crítico Resumen Es necesario entender cómo afecta la endogamia a las especies en peligro que se encuentran en programas de reproducción para la conservación para la recuperación exitosa de estas especies. El cuervo hawaiano ('Alala) (Corvus hawaiiensis) es una de las especies de ave que se encuentra en mayor peligro de extinción en todo el mundo. Esta especie se extinguió en vida libre en el 2002 y no fue hasta los recientes esfuerzos de liberación que iniciaron en 2016 que casi toda la población permaneció bajo cuidado humano en programas de reproducción para la conservación. Usamos los coeficientes de linaje endogámico (F) para evaluar los efectos de la endogamia sobre el éxito reproductivo y de supervivencia de la descendencia de los cuervos hawaianos. Usamos un análisis de árbol de regresión para identificar el nivel de endogamia (es decir, el umbral de endogamia) que explicara una disminución sustancial en la supervivencia de la descendencia de 'Alala hasta el reclutamiento. Similar a un estudio previo de la endogamia en los 'Alala, descubrimos que la endogamia tuvo un impacto negativo sobre la supervivencia de la descendencia pero que la incubación parental (vs. la artificial) de huevos mejoró la supervivencia de la descendencia hasta el reclutamiento. Además, encontramos que la endogamia no afectó de manera importante al éxito reproductivo de la descendencia, esto basado en la suposición de que los descendientes que sobreviven hasta la edad adulta se reproducen con parejas cuya relación familiar es lejana. Nuestra aplicación novedosa del análisis de árbol de regresión mostró que la descendencia con niveles de endogamia que exceden F = 0.098 tuvieron 69% menos de probabilidad de sobrevivir hasta el reclutamiento que aquella progenie con menor endogamia, lo que proporciona un valor específico de umbral para el manejo continuo de la población. Nuestros resultados resaltan la importancia de la evaluación de la depresión endogámica en todas las etapas de la historia de vida, confirman la importancia de priorizar la incubación parental de huevos sobre la artificial en los programas de conservación de aves y demuestran la utilidad del análisis de árbol de regresión como herramienta para identificar los umbrales de endogamia, cuando presentes, en cualquier población manejada por linaje.

Fecal Viral Diversity of Captive and Wild Tasmanian Devils Characterized Using Virion-Enriched Metagenomics and Metatranscriptomics.

The Tasmanian devil is an endangered carnivorous marsupial threatened by devil facial tumor disease (DFTD). While research on DFTD has been extensive, little is known about viruses in devils and whether any are of potential conservation relevance for this endangered species. Using both metagenomics based on virion enrichment and sequence-independent amplification (virion-enriched metagenomics) and metatranscriptomics based on bulk RNA sequencing, we characterized and compared the fecal viromes of captive and wild devils. A total of 54 fecal samples collected from two captive and four wild populations were processed for virome characterization using both approaches. In total, 24 novel marsupial-related viruses, comprising a sapelovirus, astroviruses, rotaviruses, picobirnaviruses, parvoviruses, papillomaviruses, polyomaviruses, and a gammaherpesvirus, were identified, as well as known mammalian pathogens such as rabbit hemorrhagic disease virus 2. Captive devils showed significantly lower viral diversity than wild devils. Comparison of the two virus discovery approaches revealed substantial differences in the number and types of viruses detected, with metatranscriptomics better suited for RNA viruses and virion-enriched metagenomics largely identifying more DNA viruses. Thus, the viral communities revealed by virion-enriched metagenomics and metatranscriptomics were not interchangeable and neither approach was able to detect all viruses present. An integrated approach using both virion-enriched metagenomics and metatranscriptomics constitutes a powerful tool for obtaining a complete overview of both the taxonomic and functional profiles of viral communities within a sample.IMPORTANCE The Tasmanian devil is an iconic Australian marsupial that has suffered an 80% population decline due to a contagious cancer, devil facial tumor disease, along with other threats. Until now, viral discovery in this species has been confined to one gammaherpesvirus (dasyurid herpesvirus 2 [DaHV-2]), for which captivity was identified as a significant risk factor. Our discovery of 24 novel marsupial-associated RNA and DNA viruses, and that viral diversity is lower in captive than in wild devils, has greatly expanded our knowledge of gut-associated viruses in devils and provides important baseline information that will contribute to the conservation and captive management of this endangered species. Our results also revealed that a combination of virion-enriched metagenomics and metatranscriptomics may be a more comprehensive approach for virome characterization than either method alone. Our results thus provide a springboard for continuous improvements in the way we study complex viral communities.

From reference genomes to population genomics: comparing three reference-aligned reduced-representation sequencing pipelines in two wildlife species.

BACKGROUND: Recent advances in genomics have greatly increased research opportunities for non-model species. For wildlife, a growing availability of reference genomes means that population genetics is no longer restricted to a small set of anonymous loci. When used in conjunction with a reference genome, reduced-representation sequencing (RRS) provides a cost-effective method for obtaining reliable diversity information for population genetics. Many software tools have been developed to process RRS data, though few studies of non-model species incorporate genome alignment in calling loci. A commonly-used RRS analysis pipeline, Stacks, has this capacity and so it is timely to compare its utility with existing software originally designed for alignment and analysis of whole genome sequencing data. Here we examine population genetic inferences from two species for which reference-aligned reduced-representation data have been collected. Our two study species are a threatened Australian marsupial (Tasmanian devil Sarcophilus harrisii; declining population) and an Arctic-circle migrant bird (pink-footed goose Anser brachyrhynchus; expanding population). Analyses of these data are compared using Stacks versus two widely-used genomics packages, SAMtools and GATK. We also introduce a custom R script to improve the reliability of single nucleotide polymorphism (SNP) calls in all pipelines and conduct population genetic inferences for non-model species with reference genomes. RESULTS: Although we identified orders of magnitude fewer SNPs in our devil dataset than for goose, we found remarkable symmetry between the two species in our assessment of software performance. For both datasets, all three methods were able to delineate population structure, even with varying numbers of loci. For both species, population structure inferences were influenced by the percent of missing data. CONCLUSIONS: For studies of non-model species with a reference genome, we recommend combining Stacks output with further filtering (as included in our R pipeline) for population genetic studies, paying particular attention to potential impact of missing data thresholds. We recognise SAMtools as a viable alternative for researchers more familiar with this software. We caution against the use of GATK in studies with limited computational resources or time.

Genomic insights into a contagious cancer in Tasmanian devils.

The Tasmanian devil faces extinction due to a contagious cancer. Genetic and genomic technologies revealed that the disease arose in a Schwann cell of a female devil. Instead of dying with the original host, the tumour was passed from animal to animal, slipping under the radar of the immune system. Studying the genomes of the devil and the cancer has driven our understanding of this unique disease. From characterising immune genes and immune responses to studying tumour evolution, we have begun to uncover how a cancer can be 'caught' and are using genomic data to manage an insurance population of disease-free devils for the long-term survival of the species.

Population demography and heterozygosity-fitness correlations in natural guppy populations: An examination using sexually selected fitness traits.

Heterozygosity-fitness correlations (HFCs) have been examined in a wide diversity of contexts, and the results are often used to infer the role of inbreeding in natural populations. Although population demography, reflected in population-level genetic parameters such as allelic diversity or identity disequilibrium, is expected to play a role in the emergence and detectability of HFCs, direct comparisons of variation in HFCs across many populations of the same species, with different genetic histories, are rare. Here, we examined the relationship between individual microsatellite heterozygosity and a range of sexually selected traits in 660 male guppies from 22 natural populations in Trinidad. Similar to previous studies, observed HFCs were weak overall. However, variation in HFCs among populations was high for some traits (although these variances were not statistically different from zero). Population-level genetic parameters, specifically genetic diversity levels (number of alleles, observed/expected heterozygosity) and measures of identity disequilibrium (g2 and heterozygosity-heterozygosity correlations), were not associated with variation in population-level HFCs. This latter result indicates that these metrics do not necessarily provide a reliable predictor of HFC effect sizes across populations. Importantly, diversity and identity disequilibrium statistics were not correlated, providing empirical evidence that these metrics capture different essential characteristics of populations. A complex genetic architecture likely underpins multiple fitness traits, including those associated with male fitness, which may have reduced our ability to detect HFCs in guppy populations. Further advances in this field would benefit from additional research to determine the demographic contexts in which HFCs are most likely to occur.

Reciprocal translocation of small numbers of inbred individuals rescues immunogenetic diversity.

Genetic rescue can reduce inbreeding depression and increase fitness of small populations, even when the donor populations are highly inbred. In a recent experiment involving two inbred island populations of the New Zealand South Island robin, Petroica australis, reciprocal translocations improved microsatellite diversity and individual fitness. While microsatellite loci may reflect patterns of genome-wide diversity, they generally do not indicate the specific genetic regions responsible for increased fitness. We tested the effectiveness of this reciprocal translocation for rescuing diversity of two immunogenetic regions: Toll-like receptor (TLR) and major histocompatibility complex (MHC) genes. We found that the relatively small number of migrants (seven and ten per island) effectively brought the characteristic TLR gene diversity of each source population into the recipient population. However, when migrants transmitted TLR alleles that were already present at high frequency in the recipient population, it was possible for offspring of mixed heritage to have decreased gene diversity compared to recipient population diversity prior to translocation. In contrast to TLRs, we did not observe substantial changes in MHC allelic diversity following translocation, with limited evidence of a decrease in differentiation, perhaps because most MHC alleles were observed at both sites prior to the translocation. Overall, we conclude that small numbers of migrants may successfully restore the diversity of immunogenetic loci with few alleles, but that translocating larger numbers of animals would provide additional opportunity for the genetic rescue of highly polymorphic immunity regions, such as the MHC, even when the source population is inbred.

Pedigree analysis reveals a generational decline in reproductive success of captive Tasmanian devil (Sarcophilus harrisii): implications for captive management of threatened species.

Captive breeding programs are an increasingly popular tool to augment the conservation of threatened wild populations. Many programs keep detailed pedigrees, which are used to prescribe breeding targets to meet demographic and genetic goals. Annual breeding targets are based on previous productivity, but do not account for changes in reproductive success that may occur over generations in captivity and which may impair the ability of a program to meet its goals. We utilize a large studbook from the Tasmanian devil (Sarcophilus harrisii) captive breeding program to investigate biological, genetic, and environmental factors that affect variation in reproductive success among individuals and over generations of captive breeding. Reproductive success declined with increasing generations in captivity: wild-born females had a 56.5% chance of producing a litter compared to a 2.8% chance for generation 5 captive-born females (N = 182) and when they did, wild-born females produced more offspring (3.1 joeys, 95% CI: 2.76-3.38, compared to 2.7 joeys, 95% CI: 2.55-2.90, in captive-born females [N = 105]). Reproductive success also declined as dam age at first breeding increased. Our results reveal a conflict with the widely cited conservation strategy to limit opportunity for selection by extending generation length through delaying reproduction, as captive breeding programs that delay female breeding with this goal in mind risk reduced productivity. Our data demonstrate the benefit of pedigree analysis to identify biological processes that reveal crucial trade-offs with conservation best-practice.

Heterogeneity in ecological and evolutionary meta-analyses: its magnitude and implications.

Meta-analysis is the gold standard for synthesis in ecology and evolution. Together with estimating overall effect magnitudes, meta-analyses estimate differences between effect sizes via heterogeneity statistics. It is widely hypothesized that heterogeneity will be present in ecological/evolutionary meta-analyses due to the system-specific nature of biological phenomena. Despite driving recommended best practices, the generality of heterogeneity in ecological data has never been systematically reviewed. We reviewed 700 studies, finding 325 that used formal meta-analysis, of which total heterogeneity was reported in fewer than 40%. We used second-order meta-analysis to collate heterogeneity statistics from 86 studies. Our analysis revealed that the median and mean heterogeneity, expressed as I2 , are 84.67% and 91.69%, respectively. These estimates are well above "high" heterogeneity (i.e., 75%), based on widely adopted benchmarks. We encourage reporting heterogeneity in the forms of I2 and the estimated variance components (e.g., τ2 ) as standard practice. These statistics provide vital insights in to the degree to which effect sizes vary, and provide the statistical support for the exploration of predictors of effect-size magnitude. Along with standard meta-regression techniques that fit moderator variables, multi-level models now allow partitioning of heterogeneity among correlated (e.g., phylogenetic) structures that exist within data.

Comparison of beak and feather disease virus prevalence and immunity-associated genetic diversity over time in an island population of red-crowned parakeets.

Pathogen outbreaks in the wild can contribute to a population's extinction risk. Concern over the effects of pathogen outbreaks in wildlife is amplified in small, threatened populations, where degradation of genetic diversity may hinder natural selection for enhanced immunocompetence. Beak and feather disease virus (BFDV) was detected for the first time in an island population of red-crowned parakeets (Cyanoramphus novaezelandiae) in 2008 on Little Barrier Island (Hauturu-o-Toi) of New Zealand. By 2013, the prevalence of the viral infection had significantly decreased within the population. We tested whether the population of red-crowned parakeets showed a selective response to BFDV, using neutral microsatellite and two immunity-associated genetic markers, the major histocompatibility complex (MHC) and Toll-like receptors (TLRs). We found evidence for selection at viral-associated TLR3; however, the ability of TLR3 to elicit an immune response in the presence of BFDV warrants confirmation. Alternatively, because red-crowned parakeet populations are prone to fluctuations in size, the decrease in BFDV prevalence over time may be attributed to the Little Barrier Island population dropping below the density threshold for viral maintenance. Our results highlight that natural processes such as adaptation for enhanced immunocompetence and/or density fluctuations are efficient mechanisms for reducing pathogen prevalence in a threatened, isolated population.

Selection on MHC class II supertypes in the New Zealand endemic Hochstetter's frog.

BACKGROUND: The New Zealand native frogs, family Leiopelmatidae, are among the most archaic in the world. Leiopelma hochstetteri (Hochstetter's frog) is a small, semi-aquatic frog with numerous, fragmented populations scattered across New Zealand's North Island. We characterized a major histocompatibility complex (MHC) class II B gene (DAB) in L. hochstetteri from a spleen transcriptome, and then compared its diversity to neutral microsatellite markers to assess the adaptive genetic diversity of five populations ("evolutionarily significant units", ESUs). RESULTS: L. hochstetteri possessed very high MHC diversity, with 74 DAB alleles characterized. Extremely high differentiation was observed at the DAB locus, with only two alleles shared between populations, a pattern that was not reflected in the microsatellites. Clustering analysis on putative peptide binding residues of the DAB alleles indicated four functional supertypes, all of which were represented in 4 of 5 populations, albeit at different frequencies. Otawa was an exception to these observations, with only two DAB alleles present. CONCLUSIONS: This study of MHC diversity highlights extreme population differentiation at this functional locus. Supertype differentiation was high among populations, suggesting spatial and/or temporal variation in selection pressures. Low DAB diversity in Otawa may limit this population's adaptive potential to future pathogenic challenges.

Development of a SNP-based assay for measuring genetic diversity in the Tasmanian devil insurance population.

BACKGROUND: The Tasmanian devil (Sarcophilus harrisii) has undergone a recent, drastic population decline due to the highly contagious devil facial tumor disease. The tumor is one of only two naturally occurring transmissible cancers and is almost inevitably fatal. In 2006 a disease-free insurance population was established to ensure that the Tasmanian devil is protected from extinction. The insurance program is dependent upon preserving as much wild genetic diversity as possible to maximize the success of subsequent reintroductions to the wild. Accurate genotypic data is vital to the success of the program to ensure that loss of genetic diversity does not occur in captivity. Until recently, microsatellite markers have been used to study devil population genetics, however as genetic diversity is low in the devil and potentially decreasing in the captive population, a more sensitive genotyping assay is required. METHODS: Utilising the devil reference genome and whole genome re-sequencing data, we have identified polymorphic regions for use in a custom genotyping assay. These regions were amplified using PCR and sequenced on the Illumina MiSeq platform to refine a set a markers to genotype the Tasmanian devil insurance population. RESULTS: We have developed a set of single nucleotide polymorphic (SNP) markers, assayed by amplicon sequencing, that provide a high-throughput method for monitoring genetic diversity and assessing familial relationships among devils. To date we have used a total of 267 unique SNPs within both putatively neutral and functional loci to genotype 305 individuals in the Tasmanian devil insurance population. We have used these data to assess genetic diversity in the population as well as resolve the parentage of 21 offspring. CONCLUSIONS: Our molecular data has been incorporated with studbook management practices to provide more accurate pedigree information and to inform breeding recommendations. The assay will continue to be used to monitor the genetic diversity of the insurance population of Tasmanian devils with the aim of reducing inbreeding and maximizing success of reintroductions to the wild.

Lack of genetic diversity across diverse immune genes in an endangered mammal, the Tasmanian devil (Sarcophilus harrisii).

The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction due to the spread of devil facial tumour disease. Polymorphisms in immune genes can provide adaptive potential to resist diseases. Previous studies in diversity at immune loci in wild species have almost exclusively focused on genes of the major histocompatibility complex (MHC); however, these genes only account for a fraction of immune gene diversity. Devils lack diversity at functionally important immunity loci, including MHC and Toll-like receptor genes. Whether there are polymorphisms at devil immune genes outside these two families is unknown. Here, we identify polymorphisms in a wide range of key immune genes, and develop assays to type single nucleotide polymorphisms (SNPs) within a subset of these genes. A total of 167 immune genes were examined, including cytokines, chemokines and natural killer cell receptors. Using genome-level data from ten devils, SNPs within coding regions, introns and 10 kb flanking genes of interest were identified. We found low polymorphism across 167 immune genes examined bioinformatically using whole-genome data. From this data, we developed long amplicon assays to target nine genes. These amplicons were sequenced in 29-220 devils and found to contain 78 SNPs, including eight SNPS within exons. Despite the extreme paucity of genetic diversity within these genes, signatures of balancing selection were exhibited by one chemokine gene, suggesting that remaining diversity may hold adaptive potential. The low functional diversity may leave devils highly vulnerable to infectious disease, and therefore, monitoring and preserving remaining diversity will be critical for the long-term management of this species. Examining genetic variation in diverse immune genes should be a priority for threatened wildlife species. This study can act as a model for broad-scale immunogenetic diversity analysis in threatened species.

Impacts of early viability selection on management of inbreeding and genetic diversity in conservation.

Maintaining genetic diversity is a crucial goal of intensive management of threatened species, particularly for those populations that act as sources for translocation or re-introduction programmes. Most captive genetic management is based on pedigrees and a neutral theory of inheritance, an assumption that may be violated by selective forces operating in captivity. Here, we explore the conservation consequences of early viability selection: differential offspring survival that occurs prior to management or research observations, such as embryo deaths in utero. If early viability selection produces genotypic deviations from Mendelian predictions, it may undermine management strategies intended to minimize inbreeding and maintain genetic diversity. We use empirical examples to demonstrate that straightforward approaches, such as comparing litter sizes of inbred vs. noninbred breeding pairs, can be used to test whether early viability selection likely impacts estimates of inbreeding depression. We also show that comparing multilocus genotype data to pedigree predictions can reveal whether early viability selection drives systematic biases in genetic diversity, patterns that would not be detected using pedigree-based statistics alone. More sophisticated analysis combining genomewide molecular data with pedigree information will enable conservation scientists to test whether early viability selection drives deviations from neutrality across wide stretches of the genome, revealing whether this form of selection biases the pedigree-based statistics and inference upon which intensive management is based.